Multi-drug resistant gram-negative bacteria (MDR-GNB) present a significant threat to successful antimicrobial chemotherapy. Among MDR-GNB, Acinetobacter baumannii that are resistant to cephalosporins, carbapenems, and quinolones are becoming a global problem. Our analysis of MDR A. baumannii isolates recovered from military medical facilities treating civilian and military personnel injured in Iraq/Kuwait (Operation Iraqi Freedom) and Afghanistan (Operation Enduring Freedom) showed that the Acinetobacter derived cephalosporinase (ADC), OXA-23 and OXA-58 like carbapenemases are the major reasons for ceftazidime and imipenem resistance. We learned that: 1) ADC is a unique cephalosporinase among the class C enzymes;2) efflux pumps contribute significantly to quinolone resistance;and 3) the regulation of the AdeABC efflux pump is central to the expression of the MDR phenotype in A. baumannii. The pressing challenge is to understand the genetic and amino acid sequence requirements for beta-lactam resistance, kinetic properties and atomic structures of the ADC and OXA carbapenemases, and the contribution of the efflux pump in MDR A. baumannii. The aims of this proposal are: 1) to determine the atomic structure of the ADC-7 beta-lactamase and to test novel boronic acid and sulfone transition state inhibitors in order to reveal important structure activity relationships that will guide new inhibitor design;2) to explore the amino acid requirements for imipenem hydrolysis by OXA carbapenemases and to determine the structure of OXA-23;3) to elucidate the role of quorum sensing and biofilm formation in MDR A. baumannii;and 4) to determine the genetic variability of the regulatory region (adeR-adeS) of the efflux pump (AdeABC) of MDR isolates by DNA sequencing. We will correlate our sequence analysis of adeR-adeS with a rapid automated PCR based electrospray ionization mass spectrometry method linked with base composition analysis that can detect mutations. Accomplishing these goals will achieve an unprecedented understanding of the protein and substrate interactions and genetic properties responsible for the MDR phenotype. This work will also serve as a paradigm for the rapid diagnosis and prediction of novel MDR phenotypes in A. baumannii, form a basis for the evaluation of novel beta-lactams, and show how enzyme drug targets influence substrate affinity and catalysis.